Selective carbon adsorption of impurities from acidic streptomycin solutions



f UNITEDfY sTA'rE 'cine 55, 66- 69 (1944); tion,-the streptomycin wasrecovered from the cul- Patented Sept. 12, 1950 PATENT OF-FIGE YSELECTIVEUARBON ABSORPTION oFIM- PURITIES FROM ACIDIC STREPTO- :MYCINSOLUTIONS Robert D. Babson and Max Tishler, Railway; N. J., assignors toMerck & (10., Ino., Rahway, N. J., a corporation of New Jersey NoDrawing. Application June 8, 1946, Serial No. 675,323

,.7 Claims. (c1. 260-210) This invention relates to processes forpreparing streptomycin and particularly to new procedures for removingimpurities from. aqueous solutions containing streptomycin. Theproduction of streptomycin by cultivation of strains of the organismActz'nomyces griseus in suitable culture medium was first reported bySchatz, Bugie, and Waksinan in Proceedings of the Society forExperimental Biology and Medi- According tothis publicature broth byadsorbing it on activated charcoal (at neutrality, eluting with dilutemineral acid followed by neutralizing the eluate, drying, and treatingto remove inorganicmatter.

. In accordance with an improvedprocedure of 2. Regarded in certain ofitsbroader aspects, the novel process of the present invention"comprises acidifying an aqueous streptomycin solutiontofa pH of about1.5 to 5.0, treating theacid solution thus formed with activatedcharcoal, filtering off the activated charcoal andv adsorbed impurities,and recovering purified streptomycin from the filtrate.

In carrying out the processof the presentinvention, the aqueousstreptomycin solution can be adjusted to a pH of 1.5 to,5.0', andpreferably to a pH of about 2.2,. by the addition of any strong one ofour colleagues A. Walti, which is fully disi closed in his pendingapplication Serial No. 577,143, filed February 9, 1945,,now Patent No.2,481,267, streptomycin, adsorbed on activated carboxylic acid. In thisprocedure the difliculties and losses encountered in separatinginorganic salts are overcome.

In practice, both of the procedures referred to above are found tobeunsatisfactory in that ex- ..c'essive amounts of organic impuritiesare adsorbed and eluted, together with the streptomycin.

These impurities hamper subsequent working up .of the streptomycin for.therapeutic uses by re- 'ducing'the potency of and imparting toxicity tothe product.

.It is now discovered, in accordance withthe present invention, thatconsiderable amounts of .at an acid pH, i. e., pH 1 to 4, with activatedcharcoal. By thistreatment, impurities are adsorbed upon the charcoalwhile streptomycin remains in 1 solution, (At pHs above 4 somestreptomycin will be lost by adsorption onthe charcoal.)

Various streptomycin solutions can be treated in this I manner includingoriginal filtered streptomycin 1 culture broth,acid eluates obtainedbyprocedures mentioned above, and other aqueous streptomycin.-.solutions in various stages of purification. The

removal of impurities from streptomycinculture broth by charcoaltreatment under acid conditions is preferably followed by. adsorption ofstreptomycin on activated charcoal under neutral conditions, and elutionwith aqueous or aqueous-alcoj holic acid, in which. lattertreatmentcertain ad- ..ditional impurities are removed in the I neutralliquor and washings priorto elution.

charcoal, is eluted hy-treating alow normality, aqueous-alcoholicsolution of a lower aliphatic organic impurities can be removed, anda-product [of increased potency canbe-produced in better yield bytreating streptomycin in aqueous solution non-oxidizing mineral ororganic carboxylic acid, Thus, hydrochloric acid, sulfuric acid,phosphoric acid, formic acid, acetic acid, and propionic acid can beusedwhile an oxidizing acid such as nitric acis is not satisfactory.

To the acidified solution is added about 1 to 5% (by weight) ofactivated charcoal and the mixture is stirred for about'l5 minutes. Theactivated charcoal is then filtered oil and washed with water,preferably using an amount of water equal to about one-fourththe volumeof the original solution. The charcoal filteitcake, containingimpurities and an insignificant amount of streptomycin, is-discarded.

The combined filtrate and washings can be worked up in various ways torecover solid streptomycin. If the starting solution is an acid eluatefrom one of the former processes above mentioned, or is a partiallypurified aqueous solution of streptomycin obtained by other means, the

solid streptomycin can berecovered by concentrating the combinedfiltrate and washings to small volume (0.1 to 0.3% of theoriginalvolume) by heating in vacuo at about 50 0., adding to the volume 'ofconcentrate thus obtained about five volumes of methanol and,fiftyvolumes of acetone causing precipitation of streptomycin,separating the precipitate by centrifuging, washing the solid withacetone, and drying.

When the starting solution is a streptomycin culture broth, thecombined, filtrate and washings above mentioned are preferably worked upas follows. The pH of the solution (filtrate and washings) is adjustedto about 6.6 by addition of alkali such as 30% sodiumjhydroxide. Anyprecipitate .formed during neutralizationis filtered off and the clearfiltrate is treated with about 1 to 5% (by weight) of activatedcharcoal, the streptomycin being adsorbed; The charcoal is washed withwater, or with aqueous-alcohol, then preferably slurried with a loweraliphatic alcohol such as methanol, ethanol, or the like, and filtered.Streptomycin is then eluted from the charcoal by adding water,treatingthe mixture witha strong non-oxidizing mineral or organic aciduntil the pH remains constant at 1 to 4 and preferably at 2 about 2.2,and filtering. (If a mineral acid is used in elution'; the filtrate isneutralized to about pH 6.5). The filtrate is then concentrated to smallvolume by heating in vacuo at about 50 (1,.

and streptomycin is recovered from the concert-" trate by precipitationfrom methanol-acetone-as "1 j above described. V i

The overall recovery of active product by the process of the presentinvention isasgood as, or better than, the recovery by former methods;and the purity or potency of the product is atleast 50 to 60% greaterthan the potency of comparable products obtained by former methods.

. was washed with 250 cc. of water,

clear filtrate was treated with 20 g. of activated charcoal (Darco(3-60). The charcoal adsorbate slurred with '75 cc. of methanol, andfiltered.

The'adsorbed streptomycin was then eluted from the charcoal and workedup as in Example "I, yielding 0.31 g. of product having an activity "of250 U./mg. (Overall recovery of activity equals The present procedurecan, if desired, be followed by a further purification involving :adysorption of streptomycin on charcoal at a strongly alkalinepI-I (i. e. apH of about 8 to 11) as fully disclosed and claimedin a copendingapplication, Bittenbender and Babson, Serial No. 675,322, filed June 8,1946.,

, As difierent grades of charcoal, andfor'that matter difierent batchesof the same grade differ considerably in activity, it willbe understoodthat the amount of charcoal required, within the ranges specified, willdepend upon the activity of the particular charcoal available. It willalso be understood that the per cent byv weight of, char coal can alsobe expressed in terms of weight per volume of solution. Thus, 2% byweight of charcoal is approximately equivalent to 20 g; per liter ofsolution.

, The advantages of the new purification pro- 'cedure are demonstratedin the followingexampleswhich are givenby way of illustration and not oflimitation.

EXAMPLE I Former procedure 1000 cc. oi'streptoniyci'nculture broth(activity added 20g. or activated charcoal (Darco G-SO).

co. in vacuo at C. The streptomycin concentrate was dissolved in 5 cc.of methanol aridt'o this solution was added 50 cc. of acetone ca-usingformation of a precipitate. The precipitate was separated bycentrifuging, washed with two 10 cc. portions of acetone, and dried invacuo, yielding 0.52 g. of product having an activity of 143 U./mg.(Overall recovery of activity equals 62%.)

EXAMPLE II I 4 New procedure 1000 cc. of streptomycin culture broth(activity 121 U./cc.) was adjusted to pH 2.2 with 10-15 cc. of 85%phosphoric acid and stirred for 15 minutes with 20 g. of activatedcharcoal (Darco (3-60). The charcoal was filtered off and washed with250 cc. of water. This charcoal contains impurities and an insignificantamount of streptomycin. Thefiltrate and washings were combined andadjusted to pH 6.5 with 30% sodium hydroxide. A smallv amount. of.flocculent precipitate which separated was filtered and the EXAMPLE IIIFormer procedure 300 cc. of acid streptomycin eluate having an activityof U./cc., and obtained by elution from a charcoal adsorbate, wasconcentrated to 0.3 to 1.0 cc. in vacuo at 50 C. The streptomycinconcentrate was dissolved in methanol, precipitated by addition ofacetone, and worked up as described Example I, yielding 0.3 g. ofproduct having an activity of 90 U./mg. (Recovery of activity fromelu'ateequals 5 i EXAMPLE IV New procedure 300 cc. of acid streptomycineluate having an activity of 90 U./cc., and obtained byeluation from acharcoal adsorbate, was adjusted to pH 2.2 with formic acid and treatedwith 15 s. of

activated charcoal (Darco G 60). The mixture was stirred for about 15minutes and the charcoal was filtered oil and washed with about 80 cc.of water. The charcoal contains impurities and an insignificant amountof streptomycin. The

combined filtrate and washings were concentrated to 0.3 to 1.0 cc. invacuo at 50 (Land the streptomycin concentrate was dissolved inmethanol, precipitated by addition of acetone, and worked up as inExample I, yielding 0.203s. of product having an activity of 133 "CL/mg.(Recovery of activityfrom eluate equals 100%.)

EXAMPLE v New procedure 2000 cc. of streptomycin culturebroth (activity72 U./mg.) was adjusted to pH 2.2 with 15525 cc. of phosphoric acid andstirred for 15 minutes with 40 g. of activated charcoal '(Darco (E -60The charcoal was filtered off and washed with 50 cc. of water. Thischarcoal, containing mainly impurities, was discarded. The filtrate andwashings were combined and adjusted to pH 6.6 with 30% sodium hydroxide,and filtered to remove a small amount of fiocculent precipitate, whichseparated. The clear filtrate was treated with 30 .The charcoal wasfiltered, .washed with 500 cc.,oi water, and slurried with cc. ofmethanol. After filtering, the charcoal was suspended in about 150 cc.of water. acidified to pH 3.0. with hydrochloric acid to elute theabsorbed streptomycin. The mixture was then filtered and the charcoalwas washed with about 5 0 cc. "of water.

The combined filtrate and washing's were neutralized to pH6.5,,concentrated to about 20c. in vacuo at 50 (I. The streptomycinconcentrate was dissolved in methanol and acetone was then added,forming a precipitate which was worked up as in Example I, yielding 0.41g. of product having an activity of 244 U./rhg. (Overall recovery ofactivity equals 69%.)

Modifications of the foregoing procedures can ;be made without departingfrom the spiritfand scope of the present invention, and'we are to be 75limited only by the appended claims.

We claim: I

1. The process that comprises acidifying an aqueous streptomycinsolution to a pH of about 1.5 to 5.0, treating the acid solution thusformed with activated charcoal, filtering ofi the activated charcoal andadsorbed impurities, and recovering purified streptomycin from thefiltrate.

2. The process that comprises acidifying a streptomycin culture broth toa pH of about 1.5 to 5.0, treating the acid solution thus formed withactivated charcoal, filtering off the. activated charcoal and adsorbedimpurities, and recovering purified streptomycin from the filtrate.

3. The process that comprises acidifying a streptomycin eluate obtainedby elution' of a charcoal adsorbate to a pH of about 1.5 to 5.0,treating the acid solution thus formed with activated charcoal,filtering off the activated charcoal and adsorbed impurities, andrecovering purified streptomycin from the filtrate.

4. The process that comprises acidifying an aqueous streptomycinsolution to a pH of about 2.2, treating the acid solution thus formedwith activated charcoal, filtering off .the activated charcoal andadsorbed impurities, and recovering purified streptomycin from thefiltrate.

5. In the purification of streptomycin, the steps that compriseadjusting the pH of an aqueous streptomycin solution to about 2.2 byaddition of a strong non-oxidizing acid, thoroughly mixing with the acidsolution about a 2 to 5% by Weight about 6.6 by addition of. alkali,thoroughly mixof activated charcoal, filtering oil the charcoal 6 withthe neutralized solution about 2% by weight of activated charcoal,thereby adsorbing streptomycin on the charcoal, filtering off thecharcoal adsorbate and eluting streptomycin from the charcoal adsorbateby treating with an aqueous solution of a strong non-oxidizing acid ata, pH of about 1 to 4, and recovering purified streptomycin from theeluate.

7. A process for recovering streptomycin of high potency from astreptomycin culture broth that comprises adjusting the pH of theculture broth to about 2.2 by addition of a strong nonoxidizing acid,thoroughly mixing with the acid solution about2 to 5% by weight ofactivated charcoal, filtering off the charcoal and adsorbed impurities,adjusting the pH of the filtrate to ing with the neutralized solutionabout 2% by Weight of activated charcoal, thereby adsorbing streptomycinon the charcoal, filtering 011? the charcoal adsorbate and washing firstwith water and then with a lower aliphatic alcohol, eluting streptomycinfrom the charcoal adsorbate by treating with an aqueous solution of astrong REFERENCES CITED The following references are of record in thefile of this patent:

Waksman et al., Proc. Soc. ExptLfBioL, Med. 49, 207-210 (1942).

Schatz et al., Proc. Soc, Exptl. Biol., Med. 55,

J. Am. Pharm. Assoc, 34,

1. THE PROCESS THAT COMPRISES ACIDIFYING AN AQUEOUS STREPTOMYCINSOLUTION TO A PH OF ABOUT 1.5 TO 5.0, TREATING THE ACID SOLUTION THUSFORMED WITH ACTIVATED CHARCOAL, FILTERING OFF THE ACTIVATED CHARCOAL ANDADSORBED IMPURITIES, AND RECOVERING PURIFIED STREPTOMYCIN FROM THEFILTRATE.